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. 2018 Mar 29;19(5):e44722. doi: 10.15252/embr.201744722

Figure 8. The recruitment of PAI‐1 to stress granules interferes with its secretion resulting in the activation of the cyclin D1/RB pathway.

Figure 8

  1. Secreted PAI‐1 levels in supernatant obtained from senescing IDH4 cells treated with AS with or without cycloheximide (CHX) were assessed by ELISA.
  2. (left) IDH4 cells treated daily during senescence for 30 min with arsenite (AS) were supplemented or not with 200 ng/ml of recombinant PAI‐1. Cells at the PRO, PRE and SEN stages were subsequently subjected to staining for β‐galactosidase activity. Phase contrast images demonstrating β‐galactosidase staining are shown. Scale bars, 200 μm. (right) Graph represents the percentage of cells that stained positive for β‐galactosidase activity (stained blue‐green) in (left). The percentage of senescent cells in each experiment was calculated using three random fields.
  3. Cells at the PRO (left panels) and PRE (right panels) stages were fixed, permeabilized and analyzed by immunofluorescence with an antibody specific for cyclin D1. Merged panels display DAPI staining and cyclin D1 staining in order to visualize the location of nuclei in cells. Scale bars, 50 μm.
  4. (left) Western blot analysis was performed using whole‐cell extracts from PRO cells, and antibodies specific for pRb (S795) and Rb proteins. (right) ImageJ was used to quantify the levels of pRb (S795) protein, which were normalized to those of Rb protein. The graph represents the normalized pRb (S795) protein signals in the UNT condition relative to treated cells in PRO.
Data Information: (B) Data are represented as a mean (represented by −) of two independent experiments. (A, D) Data are represented as a mean of three independent experiments ± SE (error bars). *P < 0.05, ***P < 0.001 (Student's t‐test).Source data are available online for this figure.